PEPTIDE INHIBITORS FOR MEDIATING STRESS RESPONSES

INHIBITEURS PEPTIDIQUES PERMETTANT DE TRAITER DES REPONSES AU STRESS

English Abstract

The present invention relates to peptides capable of inhibiting cellular and
immune stress responses in a eukaryotic cell. The invention provides
compositions and methods for the treatment of human degenerative diseases and
inflammation, utilizing peptides recognized by monoclonal anti-DNA antibodies,
the peptides having anti-apoptotic and anti-inflammatory activity. The
invention further provides antibody molecules and uses thereof for the
isolation of such peptides.

French Abstract

La présente invention concerne des peptides capables d'inhiber des réponses cellulaires et immunes au stress dans une cellule eucaryote. Cette invention concerne également des compositions et des méthodes permettant de traiter des inflammations et des maladies dégénératives chez l'humain, au moyen de peptides reconnus par des anticorps monoclonaux anti-ADN; ces peptides présentant une activité anti-apoptotique et anti-inflammatoire. Cette invention concerne également des molécules d'anticorps ainsi que les différentes utilisations de celles-ci pour isoler de tels peptides.

Claims

WE CLAIMS:
1. A peptide comprising an epitope immunoreactive with an anti-idiotypic
antibody
directed against an anti-p53 antibody, wherein the anti-p53 antibody is
immunoreactive
with at least a part of the regulatory domain of the C-terminus of p53, and
wherein the
peptide exhibits at least one activity selected from anti-apoptotic activity
and anti-
inflammatory activity and wherein the peptide is 7-25 amino acids and
comprises an
amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8.
2. The peptide of claim 1 wherein said peptide comprises from about 7 to 12
amino acids.
3. The peptide of claim 1, wherein the anti-p53 antibody is PAb-421.
4. The peptide of claim 1, wherein the anti-idiotypic antibody is selected
from the group
consisting of ldi-1 having the CDR sequences as set forth in SEQ ID NOs: 13-18
and !di-2
having the CDR sequences as set forth in SEQ ID NOs: 19-24.
5. The peptide of claim 1 wherein the peptide is capable of down regulating
immune-
mediated stress responses.
6. The peptide of claim 1 wherein the peptide has an amino acid sequence as
set forth in
any one of SEQ ID NOS: 1-4.
7. The peptide of claim 1, wherein the peptide has an amino acid sequence
as set forth in
any one of SEQ ID NOS: 5-8.
8. The peptide of claim 1, wherein the peptide has an amino acid sequence
as set forth in
any one of SEQ ID NOS: 1 , 2 and 5.
9. An isolated peptide selected from the group consisting of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7
and 8.
10. A pharmaceutical composition comprising as the active ingredient the
peptide of any
one of claims 1-9, and a pharmaceutically acceptable carrier or diluent.
11. Use of the peptide of any one of claims 1-9 for treating a disease,
disorder or condition
selected from the group consisting of: amyotrophic lateral sclerosis (ALS),
central
nervous system injury, seizures, Alzheimer's disease, Parkinson's disease,
secondary
degeneration after trauma, stroke, CNS intoxication, glaucoma, macular
degeneration,
myocardial infarction, radiation disease, hyperthermia, hypoxia, fulminant
toxic liver,
kidney failure, infertility and a neoplastic disorder in a subject in need
thereof.
12. The use of claim 11, wherein said subject has a neoplastic disorder and
is undergoing
chemotherapy and/or radiation therapy for the treatment of cancer.
13. Use of the peptide of any one of claims 1-9 for treating an
inflammatory disease or an
autoimmune disease in a subject in need thereof.
14. The use according to claim 13, wherein the inflammatory disease or
autoimmune
disease is selected from the group consisting of: type 1 diabetes, multiple
sclerosis,
systemic lupus erythematosis, autoimmune uveitis, arthritis, systemic
inflammatory
response syndrome (SIRS), inflammatory bowel disease (IBD), adult respiratory
distress
syndrome (ARDS), psoriasis, atherosclerosis, graft rejection and graft versus
host
disease.
15. The use of claim 13, wherein the disease is multiple sclerosis.
16. The use of claim 11, wherein the disease is ALS.
39

Description

DEMANDES OU BREVETS VOLUMINEUX
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.

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PEPTIDE INHIBITORS FOR MEDIATING STRESS RESPONSES
FIELD OF THE INVENTION
The present invention relates to peptides recognized by monoclonal anti-DNA
antibodies, the peptides being capable of inhibiting cellular and immune
stress
responses in a eukaryotic cell, for the treatment of human degenerative
disorders and
inflammation.
BACKGROUND OF THE INVENTION
Apoptosis
Apoptosis, or programmed cell death, is of fundamental importance to normal
biological processes including embryogenesis, maintenance of tissue
homeostasis,
cellular development of multicellular organisms, elimination of virus-infected
cells,
and the development of the immune system (Ellis et al., 1991). It is a type of
cell
death that is fundamentally distinct from degenerative death or necrosis in
that it is an
active process of gene-directed cellular self-destruction which, in some
instances,
serves a biologically meaningful homeostatic function.
p53
The p53 protein, originally identified as a tumor-associated antigen, is the
product of a tumor suppressor gene that functions to arrest the growth of
mutated or
aberrant cells (Baker et al, 1990). Functional p53 is believed to detect DNA
damage
(Lee et al, 1995) and subsequently induce DNA repair (Kastan et al, 1991),
growth
arrest (Kuerbitz et al, 1992), or apoptosis (Yonish-Rouach et al, 1991) of the
aberrant
cells. In particular, p53 controls genomic stability by eliminating
genetically damaged
cells from the cell population, and one of its major functions is to prevent
tumor
formation.
The p53 protein has at least two DNA-binding sites:
(1) the core of the p53 protein, which interacts specifically with a DNA
sequence in
the promoter region of p53 responsive genes (el-Deiry et al, 1992); and
(2) the C-terminus of the p53 protein, which can recognize features common to
damaged DNA in general (Lee et al, 1995; Foord et al, 1991).

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The p53 protein is a transcription factor that binds specifically to a
consensus
site present in the regulatory sequences of p53-dependent genes (el-Deiry et
al, 1992).
Mutation of the p53 gene in the domain encoding sequences involved in binding
to
the specific DNA regulatory site causes a loss of tumor suppression.
Therefore, it is
not surprising that a significant proportion of natural human tumors bear
mutated p53
(Hollstein et al, 1991).
p53 has a short half-life, and, accordingly, is continuously synthesized and
degraded in the cell. However, when a cell is subjected to stress, p53 is
stabilized.
Examples of cell stress that induce p53 stabilization are:
a) DNA damage, such as damage caused by UV (ultraviolet) radiation, cell
mutations, chemotherapy, and radiation therapy;
b) hyperthermia;
c) hypoxia; and
d) deregulation of microtubules caused by some chemotherapeutic drugs, e.g.,
treatment using taxol or Vinca alkaloids.
Stress-activated p53 induces a cascade of events that result in growth arrest
or
apoptosis of the stressed cell, thereby preventing the outgrowth of aberrant
cells and
tumor formation (Ko, 1996). However, excessive activation of p53 after severe
stress
can be harmful to the organism, as tissue function may be damaged by excessive
apoptosis (Komarova, 2001).
Specifically, radiation therapy and chemotherapy exhibit severe side effects,
such as severe damage to the lymphoid and hematopoietic system and intestinal
epithelia, which limit the effectiveness of these therapies. Other side
effects, like hair
loss, also are p53 mediated and further detract from cancer therapies.
Therefore, to
eliminate or reduce adverse side effects in normal tissues associated with
cancer
treatment, it would be beneficial to inhibit p53 activity in normal tissue
during
treatment of p53-deficient tumors, and thereby protect normal tissue.
Inactivation of p53 has been considered an undesirable and unwanted event,
and considerable effort has been expended to facilitate cancer treatment by
restoring
p53 function. However, p53 restoration or imitation causes the above-described
problems with respect to damaging normal tissue cells during chemotherapy or
radiation therapy. These normal cells are subjected to stress during cancer
therapy,
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which leads the p53 in the cell to cause a programmed death. The cancer
treatment
then kills both the tumor cells and the normal cells.
US Patent No. 6,593,353 discloses p53 inhibitors in the treatment of p53-
mediated diseases, conditions and injuries.
US Patent No. 6,420,136 discloses methods for modulating the activity of the
p53 protein in cells by the addition of a protein which enhances or inhibits
the
biochemical activity of p53.
US Patent No. 6,630,584 discloses a single chain antibody which recognizes
an epitope exposed on mutant, but not on wild-type p53 and a DNA molecule
encoding the single chain Fv, pharmaceutical compositions comprising the
antibody
and methods of treatment using the pharmaceutical compositions.
p53 and Stress-Associated Response
The adverse effects of p53 activity on an organism are not limited to cancer
therapies. p53 is activated as a consequence of a variety of stresses
associated with
injuries (e.g., burns) naturally occurring diseases (e.g., hyperthermia
associated with
fever, and conditions of local hypoxia associated with a blocked blood supply,
stroke,
and ischemia) and cell aging (e.g., senescence of fibroblasts), as well as a
cancer
therapy. Temporary p53 inhibition, therefore, also can be therapeutically
effective in:
(a) reducing or eliminating p53-dependent neuronal death in the central
nervous
system, i.e., brain and spinal cord injury, (b) the preservation of tissues
and organs
prior to transplanting, (c) preparation of a host for a bone marrow
transplant, and (d)
reducing or eliminating neuronal damage during seizures, for example.
In addition, various degenerative diseases, including Alzheimer's disease,
Parkinson's disease, ischemic stroke (Mattson, 2001; Martin, 2001), glaucoma
(Nickells, 1999) secondary degeneration after trauma (Raghupathi, 2000),
myocardial
infarction (Haunstetter, 1998) are associated with excessive cell death of
sensitive
tissue in response to stress. Therefore, temporary inhibition of stress-
related cell death
may serve the prevention and therapy of degenerative diseases (Komarova,
2001).
Monoclonal Antibody to the DNA-binding Domain of p53
Antibodies to DNA are characteristic of many autoimmune diseases, notably
systemic lupus erythematosus (SLE) and particularly lupus nephritis. However,
there
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is at present no generally accepted explanation for the prevalence of anti-DNA

antibodies in autoimmune disorders. Immunity to DNA appears to be driven by an

antigen (Radic et al, 1994), but self-DNA is unlikely to be the driving
antigen because
mammalian DNA usually does not induce an anti-DNA immune response (Pisetsky,
1996).
It has been reported that immunization with monoclonal antibodies can induce
immune responses that extend beyond the specificity of the antibody, probably
by
anti-idiotypic connectivity based on idiotypic determinants in the variable
regions of
the immunizing monoclonal antibody.
According to idiotypic antibody network terminology, Abl is the first
antibody, the antibody binding to the antigen, and Ab2 is the anti-idiotypic
antibody
to Abl. The variable region of Ab2 may mimic the conformation of the antigen
because both the antigen and Ab2 can be bound by Abl. Ab3 is the anti-
idiotypic
antibody to Ab2. Because of the chain of structural complementarity, Abl and
Ab3
can have similar specificity for the original antigen.
The PAb-421 antibody is a prototypic monoclonal antibody that reacts with
the C-terminal DNA-binding domain of p53. The sequences of the variable heavy
(VH) and variable light (VI) chains of the anti-p53 PAb-421 have been
elucidated (see
WO 98/56416). The use of PAb-421 antibody for the treatment of cancer was
suggested, since it activated DNA binding of p53 in vitro (see WO 94/12202).
The inventors previously reported that immunization of mice with PAb-421
induced formation of anti-idiotypic antibodies that also bind DNA (Herkel et
al.,
2000; and WO 00/23082). Two of these monoclonal anti-idiotypic antibodies,
designated Idi-1 and Idi-2, mimicked the binding properties of the p53
regulatory
domain and reacted specifically with PAb-421 and double- and single stranded
DNA.
It was suggested by the present inventors (after the priority date of the
present
invention) that damaged DNA has a chemically defined structure that is
recognized by
p53 and by Idi-1 and Idi-2 antibodies (Herkel, et al, 2004). Nowhere in the
background art was it taught or suggested that it is possible to identify
novel peptides
having anti-apoptotic and anti-inflammatory properties using such anti-
idiotypic
antibodies.
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There is an unmet need for novel compositions that may serve to attenuate
cellular and immune stress-response in normal tissue, in a manner that is
specific, safe
and effective, thereby reducing the severity of stress associated degenerative
diseases
and stress-induced inflammation.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods comprising
peptides for inhibiting cellular and immune stress-response to a variety of
stress-
associated conditions. The peptides of the present invention exhibit anti-
apoptotic and
anti-inflammatory activity, thereby increasing cell survival in cells or
tissues that are
exposed to stress.
While the use of antibodies to p53 for inducing anti-tumor immunity has been
described, the present invention demonstrates that anti idiotypic antibodies
immunoreactive with anti-p53 antibodies also may be used to define therapies
useful
to prevent or decrease cell death.
Unexpectedly, it is now disclosed that peptides, recognized by monoclonal
antibodies generated by idiotypic immunization to an anti-p53 monoclonal
antibody
are of potential use for therapy of human degenerative diseases and in
modifying
inflammatory responses. In other words, the anti-p53 antibodies (Abs) can
generate
anti-idiotypic Abs, wherein these latter Abs recognize epitopes useful for
preventing
cell death or inflammation.
The invention is based, in part, on experiments demonstrating the efficacy of
the peptides of the invention in ameliorating stress-induced cell death and
p53-
mediated response, induced by stimuli such as DNA-damaging agents,
hyperthermia,
toxic stress and y-irradiation.
Surprisingly, it was further discovered that peptides of the invention exhibit
anti-inflammatory activity, both in vitro and in vivo. Thus, the invention
demonstrates
that the peptides of the invention are useful for treating inflammatory and
autoimmune diseases.
According to a first aspect, the invention is directed to peptides comprising
an
epitope immunoreactive with an anti-idiotypic antibody directed against an
anti-p53
antibody, wherein the anti-p53 antibody is immunoreactive with at least a part
of the
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regulatory domain at the C-terminus of p53. The peptides of the invention
exhibit at
least one activity selected from anti-apoptotic activity and anti-inflammatory
activity.
In one particular embodiment, the peptides are immunoreactive with an anti-
idiotypic antibody directed against the anti-p53 antibody PAb-421 (Herkel et
al.,
2000). In other particular embodiments, the peptides are immunoreactive with
monoclonal antibodies designated Idi-1 and Idi-2, having structural mimicry
properties to the p53 regulatory domain (Herkel et al., 2004).
In one embodiment, the anti-idiotypic antibody is a molecule comprising VL-
CDR3 and VH-CDR3 sequences selected from the group consisting of: SEQ ID
NO:15 and 18 and SEQ ID NO:21 and 24. In another embodiment, the anti-
idiotypic
antibody is a molecule comprising VL regions and VII regions selected from the
group
consisting of SEQ ID NO:9 and 10, SEQ ID NO:11 and 12, analogs and derivatives

thereof.
The peptides are characterized and synthesized by methods known in the art.
In one embodiment, the peptides are characterized by mass spectrometry and
synthesized by chemical synthesis.
According to another embodiment, the peptides may have structural
complementarity to the DNA-binding domain of p53. Without wishing to be bound
to
any particular theory or mechanism of action, it is postulated that the
peptides may be
capable of binding p53 therefore preventing p53 from binding to damaged-DNA.
In
another embodiment, the peptides of the invention exhibit the activity of
binding a
protein involved in apoptosis. In another embodiment the peptides of the
invention
exhibit the activity of preventing said protein from binding to damaged DNA.
According to certain embodiments the peptide comprises a total of about 5 to
25 amino acids, preferably the peptide comprises from about 5 to about 25
amino
acids, preferably from about 7 to 12 amino acids.
In certain particular embodiments, the present invention provides novel
peptides having an amino acid sequence selected from the group consisting of
SEQ ID
NO:! to SEQ ID NO:4, analogs, derivatives or active fragments thereof having
anti-
apoptotic activity and/or anti-inflammatory activity. The peptides of the
present
invention are as follows:
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SEQ ID NO:1 - LPPLPYP, designated Stressin-1;
SEQ ID NO:2 - DLSTDALHYRTA, designated Stressin-2;
SEQ ID NO:3 - HPTNQQSLWRWP, designated Stressin-3;
SEQ ID NO:4 - SSLSVDYPTRYP, designated Stressin-4.
In other particular embodiments, the derivative is a retro-inverso peptide,
having an amino acid sequence as set forth in any one of SEQ ID NOS:5-8:
SEQ ID NO:5 ¨ PYPLPPL (all residues in the "D" isomeric form);
SEQ ID NO:6 ¨ ATRYHLADTSLD (all residues in the "D" isomeric form);
SEQ ID NO:7 ¨ PWRWLSQQNTPH (all residues in the "D" isomeric form);
SEQ ID NO:8 ¨ PYRTPYDVSLSS (all residues in the "D" isomeric form).
In one particular embodiment, the peptide has an amino acid sequence as set
forth in any one of SEQ ID NOS:1, 2 and 5.
According to other embodiments, the peptides of the present invention are
useful for selectively preventing cell death of normal tissue. In one
embodiment, the
peptides inhibit apoptotic activity of mammalian cells. In another embodiment,
the
peptides inhibit apoptotic activity of human cells.
According to certain preferred embodiments the peptides of the present
invention are capable of inhibiting apoptotic activity by at least 25%,
preferably by at
least 50%, more preferably by at least 75% and most preferably by at least
95%.
The present invention provides peptides having the ability to effectively
inhibit cellular and immune stress responses in normal tissue, and are useful
to treat a
disease or condition where inhibition of intracellular protein activity
provides a
benefit.
According to some embodiments the peptides of the invention are useful for
treating stress-associated human degenerative diseases. According to other
embodiments the peptides are capable of down regulating immune mediated stress

responses.
In other aspects, the invention is directed to an antibody molecule comprising

VL-CDR3 and VH-CDR3 sequences selected from the group consisting of: SEQ ID
NO:15 and 18 and SEQ ID NO:21 and 24, and uses thereof for the isolation of
peptides according to the invention. In one embodiment, the antibody molecule
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comprises VL regions and VH regions selected from the group consisting of SEQ
ID
NO:9 and 10 and SEQ ID NO:11 and 12.
According to yet another aspect the present invention provides a
pharmaceutical composition comprising as an active ingredient a peptide of the
invention or a salt thereof and a pharmaceutically acceptable carrier or
diluent.
According to another aspect the present invention provides a method for
modulating cellular and immune stress-associated responses in a cell of an
organism
comprising exposing the cell to an effective amount of a peptide of the
invention, an
analog, a derivative, or a salt thereof.
Diseases and inflammatory conditions that may be treated by the peptides of
the invention include, but not limited to, Alzheimer's disease, Parkinson's
disease,
secondary degeneration after trauma, stroke, CNS intoxication, glaucoma,
macular
degeneration, type 1 diabetes, multiple sclerosis, systemic lupus
erythematosis,
autoimmune uveitis, graft versus host disease, graft rejection, arthritis,
systemic
inflammatory response syndrome (SIRS) inflammatory bowel disease (IBD), adult
respiratory distress syndrome (ARDS), psoriasis, atherosclerosis, myocardial
infarction, radiation disease, hyperthermia, hypoxia, fulminant toxic liver,
kidney
failure, infertility and many others.
In another aspect, the invention provides a method for treating a degenerative
disease or condition in a subject in need thereof, comprising administering to
the
subject a therapeutically effective amount of a peptide of the invention, an
analog, a
derivative, or a salt thereof
In one embodiment, the disease or condition is a stress-associated
degenerative
disorder.
In another embodiment, said subject has a neoplastic disorder and is
undergoing chemotherapy and/or radiation therapy for the treatment of cancer.
In another embodiment, the disease or condition is selected from the group
consisting of: Alzheimer's disease, Parkinson's disease, secondary
degeneration after
trauma, stroke, CNS intoxication, glaucoma, macular degeneration, myocardial
infarction, radiation disease, hyperthermia, hypoxia, fulminant toxic liver,
kidney
failure and infertility.
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In another aspect, the invention provides a method for treating an
inflammatory disease or condition in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of a peptide
of the
invention, an analog, a derivative, or a salt thereof.
In one embodiment, the disease or condition has an etiology associated with
production of at least one pro-inflammatory cytokine selected from IL-6 and
TNF-a.
In another embodiment, the disease is an autoimmune disease.
In other embodiments, the disease or condition is selected from the group
consisting of: type 1 diabetes, multiple sclerosis, systemic lupus
erythematosis (SLE),
autoimmune uveitis, arthritis, systemic inflammatory response syndrome (SIRS)
inflammatory bowel disease (IBD), adult respiratory distress syndrome (ARDS),
psoriasis, atherosclerosis, graft rejection and graft versus host disease.
In one particular embodiment, the disease is multiple sclerosis.
In one embodiment, the peptide inhibits apoptotic activity in response to
cellular and immune stress disorders in normal tissue or cells. According to
one
embodiment the peptides of the present invention modulate intracellular
protein
activity within a cell in vivo. In another embodiment, the peptides of the
present
invention modulate intracellular protein activity within a cell ex vivo.
According to some embodiments the peptide may be administered to the
subject in need thereof by any suitable route of administration, including,
but not
limited to, orally, topically, transdermally, parenterally
These and other embodiments of the present invention will become apparent in
conjunction with the figures, description and claims that follow.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: DNA content of L12 cells treated with the DNA-damaging agent
cisplatin,
as a measure for p53-mediated cell death.
Figure 2: Cisplatin (80 M)-induced p53-mediated cell death of mouse embryo
fibroblasts.
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Figure 3: The effect of Stressin peptides on BALB/c mice when subjected to
body
irradiation at a dose of 6.5 Gy.
Figure 4: TNF-a secretion of RAW 264.7 macrophages in response to
lipopolysaccharide (LPS) or CpG-oligonucleotides is inhibited by Stressin-1.
Figure 5: Interleukin-6 secretion of RAW 264.7 macrophages in response to
lipopolysaccharide (LPS) or CpG-oligonucleotides is inhibited by Stressin-1.
Figure 6: Inhibitory effect of Stressin-1 in development of experimental
autoimmune
disease (EAE) in mice.
Figure 7: Amino acid sequences of the variable regions of PAb-421, Idi-1 and
Idi-2.
Complementarity Determining Regions (CDRs) of the light chain or of the heavy
chain are aligned.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods comprising
peptides for inhibiting cellular and immune stress-response to a variety of
stress-
associated conditions. The invention provides compositions and methods for the
treatment of human degenerative diseases and inflammation, utilizing peptides
recognized by monoclonal anti-DNA antibodies, the peptides having anti-
apoptotic
and anti-inflammatory activity.
Definitions
As used herein, the term "Linear Peptide" means a peptide or polypeptide in
which the amino acids are linked to one another via an amide bond formed
between
the alpha-amino group of one and the alpha-carboxylic group of another.
As used herein, "cell" refers to a eukaryotic cell. Typically, the cell is of
animal origin and can be a stem cell or somatic cells. Suitable animal cells
can be of,
for example, mammalian and avian origin. Examples of mammalian cells include
human, bovine, ovine, porcine, murine, rabbit cells. The cell may be an
embryonic
cell, bone marrow stem cell or other progenitor cell. Where the cell is a
somatic cell,
the cell can be, for example, an epithelial cell, fibroblast, smooth muscle
cell, blood
cell (including a hematopoietic cell, red blood cell, T-cell, B-cell, etc.),
cardiac
muscle cell, macrophage, dendritic cell, neuronal cell (e.g., a glial cell or
astrocyte).

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In the context of this invention "modulation" means inhibition; i.e., a
decrease
in expression. This modulation can be measured in ways which are routine in
the art,
for example by Western blot or ELISA assay of protein expression, or by an
hnmunoprecipitation assay of protein expression.
The term "treating" as used herein includes prophylactic and therapeutic uses,
and refers to the alleviation of symptoms of a particular disorder in a
patient, the
improvement of an ascertainable measurement associated with a particular
disorder,
or the prevention of a particular immune response (such as transplant
rejection).
Antibodies, or immunoglobulins, comprise two heavy chains linked together
by disulfide bonds and two light chains, each light chain being linked to a
respective
heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic
digestion of
an antibody yields Fv (Fragment variable) and Fc (fragment crystalline)
domains. The
antigen binding domains, Fab', include regions where the polypeptide sequence
varies. The term F(ab')2 represents two Fab' arms linked together by disulfide
bonds.
The central axis of the antibody is termed the Fe fragment. Each heavy chain
has at
one end a variable domain (VH) followed by a number of constant domains (CH).
Each light chain has a variable domain (VL) at one end and a constant domain
(CL) at
its other end, the light chain variable domain being aligned with the variable
domain
of the heavy chain and the light chain constant domain being aligned with the
first
constant domain of the heavy chain (CH1).
The variable domains of each pair of light and heavy chains form the antigen
binding site. The domains on the light and heavy chains have the same general
structure and each domain comprises four framework regions, whose sequences
are
relatively conserved, joined by three hypervariable domains known as
complementarity determining regions (CDR1-3). These domains contribute
specificity
and affinity of the antigen binding site.
The term "antibody" as used herein refers to a polypeptide ligand
substantially encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, which specifically binds and recognizes an epitope (e.g.,
an
antigen). As used herein, this term refers to intact molecules such as
polyclonal
antibodies or monoclonal antibodies (mAbs), recombinant and engineered
antibodies,
as well as to fragments thereof, such as Fab, F(ab')2, Fab miniantibodies
(see, for
,
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example, US Patent 5,910,573, US Patent 6294353, WO 96/37621, US patent
application 08/999,554), Fv, scFv (e.g. U.S. Patent Nos. 4,946,778, 5,091,513
and
5,096,815) and the like which are capable of binding the epitopic determinant.

Antibodies used in the invention can be prepared using intact polypeptides or
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal can be derived from the

translation of RNA or synthesized chemically and can be conjugated to a
carrier
protein, if desired. Commonly used carriers that are chemically coupled to
peptides
are exemplified by bovine serum albumin, thyroglobulin and keyhole limpet
hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a
mouse,
a rat, or a rabbit). Non-limitative methods of generating antibodies are
described in
the Examples hereinbelow; however, other methods well known in the art may
readily
be used.
By the term "anti-idiotypic antibody" is intended an antibody directed against
(in other words, immunoreactive with) an idiotypic determinant of another
antibody.
As used herein, the term "idiotypic determinant" refers to an antigenic
determinant or
epitope unique to the immunoglobulin product of a single clone of cells. The
idiotope
is found in the variable region of the antibody. The term "epitope" refers to
an
antigenic determinant on a molecule which is recognized by antibodies.
As used herein, the term "immunoreactive" means that the antibody is capable
of binding the antigen with a binding affinity that is indicative of an immune
reaction
to the antigen. Such affinities are well known to those of skill in the art
and include
affinities of 105 to 1014 M-1. Methods of determining the affinity of an
antibody
composition are described in Day, Advanced Immunochemistry, (2nd edition)
Wiley-
Liss, New York, N.Y. (1990).
Stressin peptides
According to a first aspect, the invention is directed to peptides comprising
an
epitope immunoreactive with an anti-idiotypic antibody directed against an
anti-p53
antibody, wherein the anti-p53 antibody is immunoreactive with at least a part
of the
regulatory domain at the C-terminus of p53. The peptides of the invention
exhibit at
least one activity selected from anti-apoptotic activity and anti-inflammatory
activity,
as will be specified hereinbelow.
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In one embodiment, the peptides are selected by anti-idiotypic monoclonal
antibodies having structural mimicry properties to the p53 regulatory domain.
According to certain embodiments, the peptides are immunoreactive with
monoclonal
antibodies raised against anti-p53 antibodies. In one particular embodiment,
the anti-
p53 antibody is PAb-421 (Herkel et al., 2000). In other particular
embodiments, the
peptides are immunoreactive with monoclonal antibodies designated Idi-1 and
Idi-2,
having structural mimicry properties to the p53 regulatory domain (Herkel et
al.,
2004).
In other embodiments, the peptides are immunoreactive with an anti-idiotypic
antibody molecule comprising VL-CDR3 and VH-CDR3 sequences selected from the
group consisting of: SEQ ID NOS:15 and 18, and SEQ ID NOS:21 and 24. In
another
embodiment, the anti-idiotypic antibody is a molecule comprising CDR sequences

selected from the group consisting of: SEQ ID NOS:13-18, and SEQ ID NO S:19-
24.
In another embodiment, the anti-idiotypic antibody is a molecule comprising VL
regions and VH regions selected from the group consisting of SEQ ID NOS:9 and
10,
SEQ ID NOS:11 and 12, analogs thereof.
The peptides are characterized and synthesized by methods known in the art.
In one embodiment, the peptides are characterized by mass spectrometry and
synthesized by chemical synthesis, as described below.
The peptides of the invention are preferably from 5 to 25 amino acids, more
preferably from 5 to 15 amino acids and most preferably from 7 to 12 amino
acids.
According to certain particular embodiment, the present invention provides
four selected peptides, designated Stressin-1 to 4 (for STress RESponse
Specific
peptide INhibitor). The amino acid sequences of certain peptides of the
invention are
listed in Table 1 and designated SEQ ID NO:1 through SEQ ID NO:4.
Unless otherwise specified, the amino acid residues described herein are
preferred to be in the "L" isomeric form. However, residues in the "D"
isomeric form
can be substituted for any L-amino acid residue, as long as the peptide
retains the
desired functional property.
In other particular embodiments, the invention provides analogs, fragments
and functional derivatives of the peptides designated SEQ ID NO:1 through SEQ
ID
NO:4, as will be described in detail hereinbelow. According to certain
particular
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embodiments, the derivatives are retro-inverso peptides having an amino acid
sequence as set forth in any one of SEQ ID NOS:5-8, as specified below. In a
particular embodiment, the peptide has an amino acid sequence as set forth in
any one
of SEQ ID NOS:1, 2 and 5.
Previously, mAbs Idi-1 and Idi-2 were demonstrated to specifically bind both
PAb-421 and DNA, single- or double-stranded (Herkel et al., 2000 of some of
the
inventors of the present invention). The present invention provides antibody
molecules directed to PAb-421 comprising variable regions selected from the
group
consisting of SEQ ID NOS:9 and 10 and SEQ ID NOS:11 and 12, and antibody
molecules comprising CDRs having amino acid sequences selected from the group
consisting of: SEQ ID NOS:13-18 and SEQ ID NOS:19-24. Such antibodies exclude
the known mAbs termed Idi-1 and Idi-2.
In one embodiment, the antibody molecule comprises VL-CDR3 and VH-
CDR3 sequences selected from the group consisting of: SEQ ID NOS:15 and 18
(corresponding to Complementarity Determining Region 3 of the light chain of
Idi-1
and Complementarity Determining Region 3 of the heavy chain of Idi-1,
respectively)
and SEQ ID NOS:21 and 24 (Idi-2). In another embodiment, the antibody molecule

comprises CDR sequences as presented in Table 4 below, having amino acid
sequences selected from the group consisting of: SEQ ID NOS:13-18 (Idi-1) and
SEQ
ID NOS:19-24 (Idi-2). In another embodiment, the antibody molecule comprises
VL
regions and VH regions (variable regions of an immunoglobulin light and heavy
chain) selected from the group consisting of SEQ ID NOS:9 and 10 (Idi-1), SEQ
ID
NOS:11 and 12 (Idi-2). The antibody molecules of the invention also include
molecules comprising analogs and derivatives of said VL regions and VH
regions, as
long as the analog or derivative is immunoreactive with the antigen-binding
portion of
PAb-421.
In another aspect, the invention provides use of antibody molecules as
described above for isolating peptides exhibiting at least one activity
selected from the
group consisting of anti-apoptotic activity and anti-inflammatory activity.
Suitable
methods utilizing these antibodies for identification and isolation of the
peptides of
the invention are disclosed hereinbelow.
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Phage display library
Phage display peptide libraries have emerged as a powerful method in
identifying such peptide agonists and antagonists. See, for example, Scott et
al.
(1990), Devlin et al. (1990), U.S. Pat. No. 5,223,409; U.S. Pat. No.
5,733,731; U.S.
Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665; U.S.
Pat. No.
5,922,545; WO 96/40987; and WO 98/15833. In such libraries, random peptide
sequences are displayed by fusion with coat proteins of filamentous phage.
Typically,
the displayed peptides are affinity-eluted against an antibody-immobilized
extracellular domain of a receptor. The retained phages may be enriched by
successive rounds of affinity purification and repropagation. The best binding
peptides may be sequenced to identify key residues within one or more
structurally
related families of peptides. See, e.g., Cwirla et al. (1997), in which two
distinct
families were identified. The peptide sequences may also suggest which
residues may
be safely replaced by alanine scanning or by mutagenesis at the DNA level.
Mutagenesis libraries may be created and screened to further optimize the
sequence of
the best binders (Lowman, 1997).
The peptides of the invention were selected and isolated from a phage display
library (Ph.D.-7 or Ph.D.-12 from New England Biolabs, Frankfurt, Germany)
with an
antibody surrogate of the p53 regulatory domain. The p53 antibody surrogate
had
been generated by idiotypic immunization to the PAb-421 monoclonal antibody
(Herkel et al., 2000) which binds the p53 regulatory domain; two monoclonal
antibodies, designated Idi-1 and Idi-2, mimicked the binding properties of the
p53
regulatory domain.
The selected candidate peptides were tested for their ability to interfere
with
the p53-mediated cellular stress response by testing their capacity to inhibit
the
response to hyperthermia of the L12 cell line (Wolf, 1984), which lacks
endogenous
p53 activity and had been stably transfected with the p53 gene or a control
vector. In
these cells, p53 activity induces growth arrest and cell survival rather than
apoptosis
in response to hyperthermia (Nitta, 1997).
Four peptides, designated Stressin-1 to -4 (for STress RESponse Specific
peptide INhibitor), were identified that at concentrations of 100 [1g/m1
inhibited p53-
mediated growth arrest after hyperthermia and induced death of almost all
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active p53, which is the response of L12 cells that lack p53 activity (Table
1); the
peptide sequences are shown in Table 2.
Alternative methods for identifying and isolating peptides
Structural analysis of protein-protein interaction may also be used to suggest
peptides that mimic the binding activity of large protein ligands. In such an
analysis,
the crystal structure may suggest the identity and relative orientation of
critical
residues of the large protein ligand, from which a peptide may be designed
(see, e.g.,
Takasaki et al., 1997). These analytical methods may also be used to
investigate the
interaction between a receptor protein and peptides selected by phage display,
which
may suggest further modification of the peptides to increase binding affinity.
Other methods compete with phage display in peptide research. A peptide
library can be fused to the carboxyl terminus of the lac repressor and
expressed in E.
coli. Another E. coli-based method allows display on the cell's outer membrane
by
fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these
and
related methods are collectively referred to as "E. coli display". In another
method,
translation of random RNA is halted prior to ribosome release, resulting in a
library of
polypeptides with their associated RNA still attached. Hereinafter, this and
related
methods are collectively referred to as "ribosome display". Other methods
employ
chemical linkage of peptides to RNA; see, for example, Roberts and Szostak
(1997).
Hereinafter, this and related methods are collectively referred to as "RNA-
peptide
screening". Chemically derived peptide libraries have been developed in which
peptides are immobilized on stable, non-biological materials, such as
polyethylene
rods or solvent-permeable resins. Another chemically derived peptide library
uses
photolithography to scan peptides immobilized on glass slides. Hereinafter,
these and
related methods are collectively referred to as "chemical-peptide screening".
Chemical-peptide screening may be advantageous in that it allows use of D-
amino
acids and other unnatural analogues, as well as non-peptide elements. Both
biological
and chemical methods are reviewed in Wells and Lowman (1992).
Phage display, in particular, is useful in generating peptides for use in the
present invention. It has been stated that affinity selection from libraries
of random
peptides can be used to identify peptide ligands for any site of any gene
product
(Dedman et al., 1993). Phage display is particularly well suited for
identifying
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peptides that bind to such proteins of interest as cell surface receptors or
any proteins
having linear epitopes (Wilson et al., 1998; Kay et al., 1998).
Synthesis of Peptides of the Invention
The peptides of the invention can be produced by any known chemical and
recombinant methods of producing an amino-acid sequence, including
peptidomimetic methodologies (Allen G., 1989; Young, 1963; Meienhofer, 1973;
Schroder and Lupke, 1965). Chemical synthesis is commonly performed by
coupling
of the amino acid residues or peptide fragments to one another in correct
order in
liquid phase to produce the desired peptide. Another common strategy is the
coupling
of the amino acids to one another starting with a solid phase (resin) to which
the C-
terminal of the last amino acid of the sequence is coupled, whereupon the C-
terminal
of the penultimate amino acid is coupled to the N-terminal of the last amino
acid, etc.,
finally releasing the built-up peptide from the solid phase (so called solid-
phase
technique).
The term "peptide" refers to molecules of 2 to 25 amino acids, with molecules
of 5 to 20 amino acids preferred and those of 7 to 12 amino acids most
preferred.
Exemplary peptides may be randomly generated by any of the methods cited
above,
carried in a peptide library (e.g., a phage display library), or derived by
digestion of
proteins.
The present invention encompasses any analog, derivative, and conjugate
containing the peptides of the invention, the amino acid sequence of which is
shown
herein so long as the peptide is capable of inhibiting apoptosis and/or
inflammation.
Thus, the present invention encompasses peptides containing non-natural amino
acid
derivatives or non-protein side chains.
The term "analog" includes any peptide or polypeptide having an amino acid
sequence substantially identical to one of the sequences specifically shown
herein in
which one or more residues have been conservatively substituted with a
functionally
similar residue and which displays the abilities as described herein. Examples
of
conservative substitutions include the substitution of one non-polar
(hydrophobic)
residue such as isoleucine, valine, leucine or methionine for another, the
substitution
of one polar (hydrophilic) residue for another such as between arginine and
lysine,
between glutamine and asparagine, between glycine and serine, the substitution
of one
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basic residue such as lysine, arginine or histidine for another, or the
substitution of
one acidic residue, such as aspartic acid or glutamic acid for another.
A peptide derivative refers to a molecule comprising the amino acid sequence
of a peptide of the invention subject to various changes, including, but not
limited to,
chemical modifications, substitutions, insertions, extensions and deletions
where such
changes do not destroy the anti-inflammatory or anti-apoptotic activity of the
peptide,
and such derivative is not a known peptide or protein. "Peptide derivative" is
intended
to include peptide mimetics, as described hereinbelow. In this regard, a
peptide of this
invention corresponds to, and preferably is identical to, one of the peptides
listed in
Table 1, where one or more changes are made so long as the polypeptide retains
the
inhibitory function of peptide of the invention in one or more of the assays
as defined
herein. With respect to the antibody molecules of the invention, a variable
region
derivative retains the ability to specifically bind (i.e. is immunoreactive
with) the
idiotypic determinant of PAb-421.
Peptide derivatives having chemical modifications include, for example,
peptides having one or more residues chemically derivatized by reaction of
side
chains or functional groups. Such derivatized molecules include, for example,
those
molecules in which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-
butyloxycarbonyl
groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types of esters or

hydrazides. Free hydroxyl groups may be derivatized to form 0-acyl or 0-alkyl
derivatives. The imidazole nitrogen of histidine may be derivatized to form N-
im-
benzylhistidine. Also included as chemical derivatives are those peptides,
which
contain one or more naturally occurring amino acid derivatives of the twenty
standard
amino acid residues. For example: 4-hydroxyproline may be substituted for
proline; 5-
hydroxylysine may be substituted for lysine; 3-methylhistidine may be
substituted for
histidine; homoserine may be substituted or serine; and ornithine may be
substituted
for lysine.
In addition, a peptide derivative can differ from the natural sequence of the
peptides of the invention by chemical modifications including, but are not
limited to,
terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by
terminal-
carboxyl-amidation, e.g., with ammonia, methylamine, and the like.
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Peptides of the present invention also include any peptide having one or more
additions and/or deletions of residues relative to the sequence of the
peptides of the
invention, the sequence of which are shown herein, so long as the requisite
inhibitory
activity on apoptosis and/or inflammation is maintained. The term "active
fragment"
thus relates to a peptide portion of a full length Stressin peptide of the
invention that
has at least one activity that is characteristic of the corresponding full-
length peptide.
Non-limitative examples of suitable methods for measuring inhibition of
apoptosis
and inflammation are demonstrated herein.
Addition of amino acid residues may be performed at either terminus of the
peptides of the invention for the purpose of providing a "linker" by which the
peptides
of this invention can be conveniently bound to a carrier. Such linkers are
usually of at
least one amino acid residue and can be of 40 or more residues, more often of
1 to 10
residues. Typical amino acid residues used for linking are tyrosine, cysteine,
lysine,
glutamic and aspartic acid, or the like.
A peptide of the invention may also be conjugated to itself or aggregated in
such a way as to produce a large complex containing the peptide. Such large
complex
may be advantageous because it has new biological properties such as longer
half-life
in circulation or greater activity.
Peptide Mimetic
Peptidomimetics are small molecules that can bind to proteins by mimicking
certain structural aspects of peptides and proteins. They are used extensively
in
science and medicine as agonists and antagonists of protein and peptide
ligands of
cellular and other receptors, and as substrates and substrate analogs for
enzymes.
A primary goal in the design of peptide mimetics has been to reduce the
susceptibility of mimics to cleavage and inactivation by peptidases. In one
approach,
one or more amide bonds have been replaced in an essentially isosteric manner
by a
variety of chemical functional groups, including, but not limited to urea
bond,
carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond.
In
another approach, a variety of uncoded or modified amino acids such as D-amino
acids and N-methyl amino acids have been used to modify mammalian peptides.
To test whether Stressin peptide may protect an organism from death by
excessive p53 activation and tissue failure by subjecting BALB/c mice to whole
body
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y-irradiation (6.5 Gy). A retro-inverso peptide was used to determine whether
prolonged in vivo half-life would give an advantage (see Example 6); retro-
inverso
peptides are resistant to proteases and consist of D-amino acids in reversed
order,
resulting in an altered peptide backbone but unchanged orientation of the side
chains
(Van Regenmortel, 1998).
As used herein, the term "retro-inverso peptide" of the Stressin-1 peptide,
for
example, as used in a variation of the invention, is intended to encompass
peptides in
which the sequence of the amino acids is reversed as compared to the sequence
in
Stressin-1 and consist of D-amino acids in reversed order.
The present invention thus provides retro-inverso Stressin peptides having an
amino acid sequence as set forth in any one of SEQ ID NOS:5-8.
The backbone can comprise a variety of atom types, including carbon,
nitrogen, oxygen, sulfur and phosphorus, with the majority of the backbone
chain
atoms typically consisting of carbon. A plurality of side chain moieties that
include a
terminal guanidino or amidino group are attached to the backbone. Although
spacing
between adjacent sidechain moieties is typically consistent, the delivery-
enhancing
transporters used in the invention can also include variable spacing between
sidechain
moieties along the backbone.
Cell Death and p53 inhibition
Apoptosis, or "programmed cell death", is a process whereby the cell executes
a "cell suicide" program. It is now thought that the apoptosis program is
evolutionarily conserved among virtually all multicellular organisms, as well
as
among all the cells_in a particular organism. Further, it is believed that in
many cases,
apoptosis may be a "default" program that must be actively inhibited in
healthy
surviving cells.
The decision by a cell to submit to apoptosis may be influenced by a variety
of
regulatory stimuli and environmental factors (Thompson, 1995). Physiological
activators of apoptosis include tumor necrosis factor (TNF), Fas ligand,
transforming
growth factor-I3, the neurotransmitters glutamate, dopamine, N-methyl-D-
aspartate,
withdrawal of growth factors, loss of matrix attachment, calcium and
glucocorticoids.
Damage-related inducers of apoptosis include heat shock, viral infection,
bacterial
toxins, the oncogenes myc, rel and El A, tumor suppressor p53, cytolytic T-
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oxidants, free radicals and nutrient deprivation (antimetabolites). Therapy-
associated
apoptosis inducers include gamma radiation, UV radiation and a variety of
chemotherapeutic drugs, including cisplatin, doxorubicin, bleomycin, cytosine
arabinoside, nitrogen mustard, methotrexate and vincristine. Toxin-related
inducers or
apoptosis include ethanol and d-amyloid peptide. Apoptosis can have
particularly
devastating consequences when it occurs pathologically in cells that do not
normally
regenerate, such as neurons. Because such cells are not replaced when they
die, their
loss can lead to debilitating and sometimes fatal dysfunction of the affected
organ.
Such dysfunction is evidenced in a number of neurodegenerative disorders that
have
been associated with increased apoptosis, including Alzheimer's disease,
Parkinson's
disease, amyotrophic lateral sclerosis, retinitis pigmentosa and cerebellar
degeneration.
In one aspect, this invention provides compositions and methods for
preventing or inhibiting apoptosis in eukaryotic cells. Irrespective of the
mechanism
by which the peptides of the invention mediates stress responses, and without
wishing
to be bound by any theory or mechanism of action, it is postulated that the
peptides
may be capable of binding p53 therefore preventing p53 to be bound to damaged-
DNA.
A potential therapeutic inhibitor of p53 is a compound that acts at any stage
of
the p53 signaling pathway, and leads to functional inactivation of a p53-
mediated
response (i.e., blocking of p53-dependent growth arrest, apoptosis, or both).
Prior
investigators did not consider therapeutic p53 inhibitors because therapeutic
p53
suppression was considered a disadvantage leading to the onset and
proliferation of
cancerous tumors. The present invention, therefore, is directed to the
therapeutic and
reversible inhibition of p53 activity, and to peptides capable of such
inhibition.
However, there are several objectives that should be mentioned before a
therapy involving suppression of p53 or any other protein that plays a role in

apoptosis or inflammation-related disorders is implemented, for example:
a) providing an inhibitor that is sufficiently efficacious in vivo for
practical
administration as a therapeutic drug;
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b) providing an inhibitor that has a sufficiently low toxicity for use in
therapy,
and also does not cause undesirable side effects at concentrations sufficient
to
inhibit p53 activity;
c) exhibiting inhibition that is reversible. Long-term p53 inactivation, for
example, can significantly increase the risk of cancer;
d) during temporary p53 inactivation, the cells should recover from the
applied
stress and the p53-activating signal should be eliminated or reduced,
otherwise
restoration of p53 activity while the p53-activating signal is active could
result
in cell damage;
e) the p53 suppression therapy is not associated with a dramatic increase in
the
frequency of cancer development.
The peptides of the invention can be used alone, or, for example, in
conjunction with chemotherapy or radiation therapy during cancer treatment, to

protect normal cells from p53 programmed death due to stresses inflicted by a
cancer
treatment or by a disease or trauma. In addition, during chemotherapy, both
tumor and
normal cells are destroyed. Tumor cells are preferentially killed compared to
normal
cells, which is the basis of a successful chemotherapy. By administering a
therapeutic
p53 inhibitor, for example, normal cells are protected, and the dose of the
chemotherapeutic agent, therefore, can be increased to more effectively treat
the
cancer.
It should be understood that the peptides of the present invention do not
necessarily act via modulating p53 activity as some of these peptides
exhibited anti-
apoptotic activity in p53-deficient cell lines and in p53 activity assays.
Methods to measure apoptosis
Apoptosis is an active, gene-directed self-destruction process of the cell and
is
associated with characteristic morphological and biochemical changes. Nuclear
and
cytoplasmic condensation and fragmentation of the dying cell into membrane-
bound
apoptotic bodies are typical characteristics of apoptosis. Another feature of
apoptotic
cell death is the chromosomal DNA degradation into oligonucleosomal fragments
after the activation of specific nucleases.
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By "inhibiting apoptosis" or "inhibits apoptotic activity" is meant any
decrease in the number of cells that undergo apoptosis relative to an
untreated control
(i.e. cells not exposed to the peptides of the invention). Preferably, the
decrease is at
least 25%, more preferably the decrease is at least 50%, and most preferably
the
decrease is at least one-fold.
Flow cytometry offers a wide variety of possibilities to measure apoptosis.
Different methods have been established and implemented, some which stain on
the
cell surface and some which stain intracellularly.
One of the first approaches was, beside the observation that apoptotic cells
shrink and have higher intracellular granularity, to stain with DNA specific
fluorochromes (e.g. propidium iodide [PI], ethidium bromide [EtBr]). As soon
as a
lethal hit is being induced, the DNA starts to change its profile. Apoptotic
DNA not
only consists of fragmented DNA (visualized as shorter bands, so called DNA
ladder,
in an agarose gel) but is also partially digested into single nucleotides, so
that
fluorochromes, like PI or EtBr, have less DNA to stain (Nicoletti et al.,
1991). This is
typically observed by a shift to the left, called sub-G1 peak, on the specific

fluorochrome detection channel in the FACScanTM (from Becton Dickinson, USA).
Another method is the terminal deoxynucleotidyl transferase (TdT)-mediated
endlabeling of the DNA strand breaks (TUNEL). The TUNEL method detects DNA
strand breaks in cells undergoing apoptosis. TdT is an enzyme which catalyzes
the
addition of deoxyribonucleotide triphosphate to the 3'-OH ends of double or
single-
stranded DNA. Unlike normal cells, apoptotic cell nuclei incorporate exogenous

nucleotides (dUTP)-DIG in the presence of TdT. An anti-DIG antibody fragment
with
a conjugated fluorochrome enables the visualization of apoptotic cells. An
increase of
apoptotic cells causes a higher number of DNA fragments and consequently a
brighter
fluorescence. An advantage of this method is the very high specificity
(Gavrieli et al.,
1992). A disadvantage of this method is that it is expensive and can only be
used for a
small set of samples, because it is time intensive. Therefore, it is not
applicable for
large screening programs.
The loss of cell membrane polarity and the presentation of increased amounts
of phosphatidyl serine (PS) on the outside of the cell membrane during the
early phase
of apoptosis has led to yet a new approach. Annexin .V is a calcium-dependent
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phospholipid binding protein with high affinity for PS. The cell membrane
integrity is
maintained in the early and intermediate phases of apoptosis. Early and
intermediate
apoptotic cells show increased binding of Annexin-FITC and are mainly negative
for
PI-staining. Late apoptotic stages and necrotic cells become double positive,
because
of PS presentation on the surface and the PI staining of intracellular nucleic
acids due
to disintegration of the membrane. This method is also costly and labor
intensive.
Other methods for measuring apoptosis in vivo and in vitro are disclosed in US

Pat Nos. 6,726,895 and 6,723,567.
Inflammatory stress responses and TNF-a and IL-6 mediated Inflammation
The mammalian response to stress includes not only the response of the
stressed cell, but also the complex activity of the immune system known as
inflammation (Nathan et al., 2002), which includes a large number of immune
activities that serve tissue maintenance and healing (Cohen, 2000).
Tumor necrosis factor (TNF) and interleukin-6 (IL-6) are important biological
entities collectively referred to as pro-inflammatory cytokines. These, along
with
several other related molecules, mediate the inflammatory response associated
with
the immunological recognition of infectious agents. The inflammatory response
plays
an important role in limiting and controlling pathogenic infections.
Elevated levels of pro-inflammatory cytokines are also associated with a
number of diseases of autoimmunity such as toxic shock syndrome, rheumatoid
arthritis, osteoarthritis, diabetes and inflammatory bowel disease (Dinarello,
et al.,
1984). In these diseases, chronic elevation of inflammation exacerbates or
causes
much of the pathophysio logy observed.
An important and accepted therapeutic approach for potential drug
intervention in these diseases is the reduction of pro-inflammatory cytokines
such as
TNF (also referred to in its secreted cell-free form as TNF-a) and IL-6. A
number of
anti-cytokine therapies are currently in clinical trials. Efficacy has been
demonstrated
with a monoclonal antibody directed against TNF-a in a number of autoirnmune
diseases (Heath, 1997). These include the treatment of rheumatoid arthritis,
Crohn's
disease and ulcerative colitis (Rankin, 1997, and Stack et al., 1997). The
monoclonal
antibody is thought to function by binding to both soluble TNF-a and to
membrane
bound TNF.
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Traumatic brain injury triggers a cascade of events resulting in delayed
edema,
necrosis and impaired function. Harmful mediators are accumulating in the
brain after
injury and recently, the role of cytokines in the pathophysiology of brain
injury has
been suggested. Spatial and temporal induction of TNF-a and IL-6 activity in
rat brain
after closed head injury has been previously reported. An inhibitor of TNF-a
production, HU-211, was shown to improve the outcome of closed brain injury in
an
experimental model (Shohami, et al., 1997). Atherosclerosis is known to have
an
inflammatory component and cytokines such as IL-1 and TNF have been suggested
to
promote the disease.
The pro-inflammatory cytokine IL-6 has been implicated with the acute phase
response. IL-6 is a growth factor in a number in oncological diseases
including
multiple myeloma and related plasma cell dyscrasias (Treon, et al., 1998,). It
has also
been shown to be an important mediator of inflammation within the central
nervous
system. Elevated levels of IL-6 are found in several neurological disorders
including
AIDS dementia complex, Alzheimer's disease, multiple sclerosis, systemic lupus
erythematosus, CNS trauma and viral and bacterial meningitis (Gruol et al.,
1997). IL-
6 also plays a significant role in osteoporosis. In murine models it has been
shown to
effect bone resorption and to induce osteoclast activity (Ershler et al.,
1997).
WO 01/01986 discloses particular compounds alleged to having the ability to
inhibit TNF-a. WO 98/52558 discloses heteroaryl urea compounds which are
indicated to be useful in treating cytokine mediated diseases. WO 99/23091
discloses
another class of urea compounds which are useful as anti-inflammatory agents.
WO
99/32463 relates to aryl ureas and their use in treating cytokine diseases and

proteolytic enzyme mediated disease.
The present invention shows that Stressin peptides are useful in interfering
and
blocking both TNF-a and IL-6 secretion by macrophage cells in response to
innate
activators such as lipopolysaccharide (LPS) and CpG oligonucleotides (see
Example
7). Therefore, these peptides are able to modify the pro-inflammatory
signaling
pathway in immune cells. As is demonstrated for Stressin-1 below, the peptides
of the
invention play a role in down-regulating the inflammatory immune response to
stress.
The present invention further demonstrates the anti-inflammatory properties of

Stressin peptides on autoimmune inflammatory diseases, as exemplified on

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experimental autoimmune encephalomyelitis (EAE), an animal model of human
multiple sclerosis (see Example 8).
Pharmaceutical compositions and therapeutic use
In another aspect, the invention relates to a pharmaceutical composition
comprising a therapeutically effective amount of a peptide of the invention
and a
pharmaceutically acceptable carrier.
As used herein a "pharmaceutical composition" refers to a preparation of one
or more of the agents described herein, or physiologically acceptable salts or
solvents
thereof, with other chemical components such as physiologically suitable
carriers and
excipients. The purpose of a pharmaceutical composition is to facilitate
administration
of a compound to an organism.
The preparation of pharmaceutical compositions, which contain peptides or
polypeptides as active ingredients is well known in the art. Typically, such
compositions are prepared as indictable, either as liquid solutions or
suspensions,
however, solid forms, which can be suspended or solubilized prior to
injection, can
also be prepared. The preparation can also be emulsified. The active
therapeutic
ingredient is mixed with inorganic and/or organic carriers, which are
pharmaceutically
acceptable and compatible with the active ingredient. Carriers are
pharmaceutically
acceptable excipients (vehicles) comprising more or less inert substances when
added
to a pharmaceutical composition to confer suitable consistency or form to the
composition. Suitable carriers are, for example, water, saline, dextrose,
glycerol,
ethanol, or the like and combinations thereof In addition, if desired, the
composition
can contain minor amounts of auxiliary substances such as wetting or
emulsifying
agents and pH buffering agents, which enhance the effectiveness of the active
ingredient.
Toxicity and therapeutic efficacy of the peptides described herein can be
determined by standard pharmaceutical procedures in cell cultures or
experimental
animals, e. g., by determining the IC50 (the concentration which provides 50%
inhibition) and the LD50 (lethal dose causing death in 50 % of the tested
animals) for a
subject compound. The data obtained from these cell culture assays and animal
studies can be used in formulating a range of dosage for use in human. The
dosage
may vary depending upon the dosage form employed and the route of
administration
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utilized. The exact formulation, route of administration and dosage can be
chosen by
the individual physician in view of the patient's condition. (See e.g., Fingl
et al.,
1975).
The amount of active agent used in an administration composition of the
present invention is an amount effective to accomplish the purpose of the
particular
active agent for the target indication. The amount of active agent in the
compositions
typically is a pharmacologically, biologically, therapeutically, or chemically
effective
amount. However, the amount can be less than that amount when the composition
is
used in a dosage unit form because the dosage unit form may contain a
plurality of
compounds or active agents in a single composition or may contain a divided
pharmacologically, biologically, therapeutically, or chemically effective
amount. The
total effective amount can then be administered in cumulative units
containing, in
total, an effective amount of the active agent.
A therapeutically effective amount of a peptide of the invention is an amount
that when administered to a patient is capable of exerting an anti-apoptotic
activity
and/or an anti-inflammatory activity. Assays for detecting the anti-apoptotic
activity
of the peptide of the invention include, but are not limited to, staining DNA
with
specific fluorochromes such as propidium iodide and ethidium bromide, Annexin
V
assays, TUNEL assays and the like; certain non-limitative examples of such
assays
are presented in the Examples below. Assays for detecting anti-inflammatory
activity
of the peptides are also well known in the art; non-limitative examples of
such
methods are presented in the Examples below.
Although an appropriate dosage of a peptide of the invention varies depending
on the administration route, age, body weight sex or conditions of the
patient, and
should be determined by the physician in the end, the dose suitable for adult
humans
can generally be between about 0.2-2000 mg/kg body weight, preferably between
about 2-200 mg/kg.
The pharmaceutical compositions of the present invention comprises one or
more compounds of the present invention, and one or more excipients or
diluents. In
one embodiment, one or more of the compounds, or solvates, or salts of these
compounds.
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The term "pharmaceutically acceptable salt" as used herein, refers to salts
which are substantially non-toxic to living organisms. Typical
pharmaceutically
acceptable salts include those salts prepared by reaction of the compounds of
the
present invention with a pharmaceutically acceptable mineral or organic acid.
Such
salts are also known as acid addition salts.
The compositions comprising the compounds and active agents have utility in
the delivery of active agents to selected biological systems and in an
increased or
improved bioavailability of the active agent compared to administration of the
active
agent without the delivery agent. Delivery can be improved by delivering more
active
agent over a period of time, or in delivering active agent in a particular
time period
(such as to effect quicker or delayed delivery) or over a period of time (such
as
sustained delivery).
Pharmaceutical compositions for use in accordance with the present invention
thus may be formulated in conventional manner using one or more
physiologically
acceptable carriers comprising excipients and auxiliaries, which facilitate
processing
of the active compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration chosen.
The pharmaceutical compositions can be administered locally or systemically
by any conventional and appropriate route including, but not limited to, oral,
intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous,
transdermal,
intrathecal, topical, rectal, buccal, inhalational or intranasal.
For injection, the compounds of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hank's
solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. Such
penetrants for example DMSO, or polyethylene glycol are generally known in the
art.
Pharmaceutical compositions, which can be used orally, include push-fit
capsules made of gelatin as well as soft, sealed capsules made of gelatin and
a
plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches,
lubricants such as talc or magnesium stearate and, optionally, stabilizers.
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In soft capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene
glycols. In
addition, stabilizers may be added. All formulations for oral administration
should be
in dosages suitable for the chosen route of administration.
Alternatively, the compounds of the present invention can be incorporated into
oral liquid preparations such as aqueous or oily suspensions, solutions,
emulsions,
syrups, or elixirs, for example. Moreover, formulations containing these
compounds
can be presented as a dry product for constitution with water or other
suitable vehicle
before use. Such liquid preparations can contain conventional additives, like
suspending agents, such as sorbitol syrup, methyl cellulose, glucose/sugar
syrup,
gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate
gel, and
hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan
monooleate, or
acacia; nonaqueous vehicles (which can include edible oils), such as almond
oil,
fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol;
and
preservatives, such as methyl or propyl p-hydroxybenzoate and sorbic acid.
For administration by inhalation, the peptides for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
a pressurized pack or a nebulizer with the use of a suitable propellant, e.
g.,
dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or
carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges
of, e.g., gelatin for use in an inhaler or insufflator may be formulated
containing a
powder mix of the peptide and a suitable powder base such as lactose or
starch.
The pharmaceutical compositions of the invention are also useful for topical
and intralesional application. As used herein, the term "topical" means
"pertaining to
a particular surface area", e.g. skin and mucosa, and the topical agent
applied to a
certain area of said surface will affect only the area to which it is applied.
The
formulations of the peptides/peptide analogs may be administered topically as
a gel,
ointment, cream, emulsion, sustained release formulation including a
transdermal
patch, and may comprise liposomes and any other pharmaceutically acceptable
carrier
suitable for administration of the drug topically. The pharmaceutical
compositions
herein described may also comprise suitable solid of gel phase carriers or
excipients.
Examples of such carriers or excipients include, but are not limited to,
calcium
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carbonate, calcium phosphate, various sugars, starches, cellulose derivatives,
gelatin
and polymers such as polyethylene glycols.
In another aspect, the invention provides a method for modulating cellular and

immune stress-associated responses in a cell of an organism comprising
exposing the
cell to an effective amount of a peptide of the invention.
In other aspects, the present invention relates to methods of treating or
preventing the symptoms of inflammatory conditions and/or degenerative
diseases
and disorders, comprising administering to a patient suffering from the
disease a
therapeutically effective amount of a peptide of the invention. Yet another
aspect of
the present invention is to provide a method of reducing or eliminating death
of
normal cells attributable to trauma or a disease comprising administering a
therapeutically effective amount of a peptide according to the invention to an

organism to inhibit stress-related protein activity.
In certain embodiments, the peptide is in the form of a pharmaceutical
composition comprising an effective amount of said peptide and a
pharmaceutically
acceptable carrier or diluent.
Stress associated responses are associated with diseases and disorders
including, for example, pathological conditions such as neurodegenerative
diseases
(e.g. stroke, Parkinson's, and Alzheimer's disease), myocardial infarction,
exposure to
radiation or chemotherapeutic agents, inflammation, injuries (e.g., burns and
central
nervous system injuries), cell aging, hyperthermia, seizures, hypoxias (e.g.,
ischemia
and stroke), and in transplant tissues and organs prior to transplanting.
These conditions also include autoimmune diseases, characterized by a state of

immunization of an individual against at least one of the body's normal
constituents.
These phenomena are observed in particular in pathologies including, but not
limited
to infections associated with SLE (Systemic Lupus Erythematosus disease),
Gougerot-Sjogren syndrome (or Sjogren's disease) and rheumatoid polyarthritis,
as
well as pathologies such as sarcoidosis and osteopenia, spondylarthritis,
scleroderma,
multiple sclerosis, amyotrophic lateral sclerosis, hyperthyroidism, Addison's
disease,
autoimmune hemolytic anemia, Crohn's disease, Goddpasture's syndrome, Graves'
disease, Hashimoto's thyroiditis, idiopathic purpural hemorrhage, insulin-
dependent
diabetes, myasthenia, pemphigus vulgaris, pernicious anemia, poststreptococcal

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glomerulonephritis, psoriasis and spontaneous sterility, as well as immediate
or
delayed phenomena observed during graft rejections and graft-versus host
disease. In
one particular embodiment, the peptides of the invention are useful for the
treatment
of multiple sclerosis, as exemplified in Example 8 herein.
The phenomenon of graft rejection is a state of immunization of an individual
against foreign constituents (bodily fluids such as blood, cerebrospinal
fluid, etc.,
cells, tissues, organs, antibodies, etc.) deliberately implanted into the
patient.
As used herein, the terms "degenerative disorder" "degenerative disease" and
"degenerative condition" are directed to any disorder, disease or condition
characterized by inappropriate cell proliferation or inappropriate cell death
or in some
cases, both, or aberrant or disregulated apoptosis. These conditions also
include
conditions in which, although appropriate and regulated at the level of a
single cell,
excessive apoptosis is associated with organ dysfunction or failure.
In one embodiment, the peptides are useful to prevent cell death in non-
malignant tissue or cells in a subject having a neoplastic disorder and
undergoing
chemotherapy and/or radiation therapy for the treatment of cancer.
The terms "inflammatory disease" and "inflammatory condition", as used
herein, mean any disease or condition in which an excessive or unregulated
inflammatory response leads to excessive inflammatory symptoms, host tissue
damage, or loss of tissue function.
In one embodiment, the inflammatory disease or condition is an autoimmune
disease. In a particular embodiment, the autoimmune disease is multiple
sclerosis.
In another embodiment, the inflammatory disease or condition has an etiology
associated with production of at least one pro-inflammatory cytokine selected
from
IL-6 and TNF-a, as discussed herein.
The following examples are to be considered merely as illustrative and non-
limiting in nature. It will be apparent to one skilled in the art to which the
present
invention pertains that many modifications, permutations, and variations may
be made
without departing from the scope of the invention.
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EXAMPLES
Example 1: Peptide selection from phage display libraries:
The monoclonal anti-PAb-421 antibodies Idi-1 and Idi-2 were generated and
characterized as described (Herkel et al., 2004). Briefly, BALB/c mice were
immunized three times with PAb-421 and the spleen cells of the mouse that
produced
the highest anti-PAb-421 titers were fused with NSO myeloma cells.
Supernatants of
the growing cells were screened by ELISA for binding to PAb-421 and to DNA.
The
hybridomas Idi-1 and Idi-2 were isolated and cloned twice by limiting
dilution.
Ph.D.-7 or Ph.D.-12 libraries from New England Biolabs, Frankfurt, Germany
were screened according to manufacturers instructions. Briefly, three rounds
of
selection by Idi-1 or Idi-2 monoclonal antibodies were performed and consensus

peptide sequences were identified by sequencing of phage DNA. Candidate
peptides
were then synthesized by Sigma-Genosis (Pampisford, UK) and further studied in
functional assays, as described hereinbelow.
Example 2: Examining the effect of candidate peptides on p53-mediated
growth arrest.
The selected candidate peptides were tested for their ability to interfere
with
the p53-mediated cellular stress response by testing their capacity to inhibit
the
response to hyperthermia of the L12 cell line (Wolf, 1984), which lacks
endogenous
p53 activity and had been stably transfected with the p53 gene or a control
vector. In
these cells, p53 activity induces growth arrest and cell survival rather than
apoptosis
in response to hyperthermia (Nitta, 1997).
The amount of cell death was assessed by staining the cells with the vital dye
trypan blue (Sigma) and counting the ratio of dead/live cells per visual field
with a
light microscope.
Results: incubation for 2 hours at 42 C induced death of all cells that lacked

p53 and, in contrast, transient growth arrest and survival of about 80% of the
cells
with active p53. Four peptides, designated Stressin-1 to -4 (for STress
RESponse
Specific peptide INhibitor), were identified that at concentrations of 100
1.ig/m1
inhibited p53-mediated growth arrest after hyperthermia and induced death of
almost
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all cells with active p53, which is the response of L12 cells that lack p53
activity
(Table 1); the peptide sequences are shown in Table 2.
Table 1: Incidence of cell death induced by hyperthermia in L12 cells with or
without active p53 and inhibition of the p53-dependent stress response by
Stressin
peptides.
no peptide Stressin 1 Stressin 2 Stressin 3 Stressin 4
L12 without p53 100% n.d. n.d. n.d. n.d.
L12 with p53 20% 100% 90% 80% 80%
Table 2: Amino acid sequences of Stressin peptides that modify the cellular
response to stress.
SEQ ID NO: Amino acid sequence Name
1 LPPLPYP Stressin-1
2 DLSTDALHYRTA Stressin-2
3 HPTNQQSLWRWP Stressin-3
4 SSLSVDYPTRYP Stressin-4
Example 3: Effect of Stressin peptides on p53-mediated cell death induced by
DNA damage.
L12 cells were treated for 48 hours with 50pM of the DNA-damaging agent
Cisplatin and p53-mediated cell death was determined by measuring the DNA
content
of cells stained with propidium iodide (Figure 1). Cells incubated in the
absence of
peptide responded to Cisplatin treatment with p53-mediated cell death.
Results: FACS analysis shows that treatment with Stressin-1 or Stressin-2
rescued 35% or 25% of the cells, respectively, from p53-mediated cell death.
Example 4: Effect of Stressin peptides on p53-mediated cell death in non-
transformed cells.
Mouse embryo fibroblasts (MEF) were treated with cisplatin (80 M) in the
presence or absence of Stressin-1 peptide as described in Example 3, and cell
death
was assessed by incorporation of the vital dye Neutral red (Sigma,
Taufkirchen,
Germany), O.D. at 540nm was read in an ELISA reader.
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The percent of viable cells is presented in Figure 2A; viability was
calculated
by the equation:
Cell viability = sample 0D540 x 100 / 0D540 of untreated cells.
Micrographs of the treated cells are presented in Figure 2B.
Figure 2 shows that Stressin-1 inhibited cell death of MEF cells induced by 80
1.1M Cisplatin in a dose-dependent way with maximal efficiency at a
concentration of
50 M.
Example 5: Effect of Stressin peptides on cell death induced by toxic stress.
To learn whether Stressin peptide may protect from toxic stress, primary
hepatocyte cultures were incubated with or without ethanol at a concentration
of
0.6%, and with 501AM Stressin-1 or without peptide. After 48 hours, the
numbers of
dead and alive cells were determined by trypan blue exclusion (Table 3).
Results: All hepatocytes exposed to ethanol died in the absence of Stressin-1;

in contrast, Stressin-1 rescued 20% hepatocytes from ethanol-induced cell
death.
Table 3: Survival of hepatocytes exposed to a lethal dose of ethanol is
promoted by Stressin-1 peptide.
Untreated Stres sin-1
Hepatocytes without ethanol 100% 100%
Hepatocytes with 0.6% ethanol 0% 20%
Example 6: Effect of Stressin peptides on mice subjected to ?-irradiation
BALB/c mice were subjected to whole body y-irradiation (6.5 Gy). One hour
after irradiation, the mice received intraperitoneally either Stressin-1 (n =
7; SEQ ID
NO:1) or a modified, retro-inverso Stressin-1 peptide (n = 7; SEQ ID NO:5),
both at a
concentration of 500m/mouse, or a sham injection with saline (n = 6). The
retro-
inverso peptide was used to determine whether prolonged in vivo-half-life
would give
an advantage.
Results: After 17 days, 66% of the sham-treated group were dead; in contrast,
none of the mice treated with the Stressin-1 peptide and only 29% of the mice
treated
with the modified Stressin-1 peptide were dead. After 40 days only 33% in the
sham
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group recovered from radiation disease; in contrast, 57% and 86% of the mice
treated
with the modified or the unmodified Stressin-1 peptide recovered from
radiation
disease (Figure 3).
Example 7: Effect of Stressin peptides on LPS- and CpG-induced cytokine
secretion.
To learn whether Stressin peptides may modify the inflammatory response to
stress signals, the response of RAW 264.7 macrophage line cells to pro-
inflammatory
microbial signals, lipopolysaccharide (LPS) and CpG oligonucleotides, was
studied.
The cells were incubated with LPS or CpG oligonucleotides in the presence or
absence of Stressin-1 (501AM). After 6 hours, the amounts of secreted TNF-a
(Figure
4) or Interleukin-6 (Figure 5) in culture supernatant, as a measure for
macrophage
activation, were determined by specific ELISA reagents and anti-TNF-a and anti-
IL-6
antibodies (R&D Systems, Wiesbaden, Germany).
Results: Stressin-1 inhibited macrophage activation and secretion of TNF-a
and Interleukin-6 induced by both LPS and CpG oligonucleotides.
Example 8: Stressin-1 protects mice from experimental autoimmune disease
(EAE).
MBP Ac1-9-specific T cell receptor-transgenic Tg4 mice (Liu et al., 1995)
were immunized subcutaneously with 200m of modified (Y at position 4) Ad -9
peptide in complete Freund's Adjuvant followed by intraperitoneal
administration of
200ng of Pertussis toxin the next day. At one hour after MBP immunization one
group of mice (n=4) received 100 1 of PBS intraperitoneally and another group
of
mice (n=6) received 500m Stressin-1 peptide in 100111 PBS intraperitoneally.
The
mice were then tested for the development of experimental autoimmune
encephalomyelitis by assessment of the clinical EAE score. As can be seen in
Figure
6, Stressin-1 protects mice from EAE.
Example 9: Sequencing of Idi-1 and Idi-2 variable regions.
Total RNA was extracted from Idi-1 and Idi-2 hybridomas using TriReagent
(Molecular Research Center, INC.), and the RNA was used as a template for cDNA
synthesis using SuperScript Reverse Transcriptase (Invitrogen, Karlsruhe,
Germany).
PCR amplification of the heavy and light chain variable regions were performed
using
primers specific for the respective flanking constant region: 5'

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CGGGAATTCCCCAGGTGCAGCTGCAGCAGTCTGG SEQ ID NO:25 and 3'
GCGGGCCCTCGAGTCTATGTACATATGCAAGGCTTACAACC SEQ ID NO:26
for the heavy chain; 5' CGCGCAAGCTTGATATTGTGATAACCCAGGATGA
SEQ ID NO:27 and 3' GATGGTGGGAAGATG SEQ ID NO:28 for the light chain.
PCR products were purified and sequenced using the same primers.
The variable regions of Idi-1 (Idi-1 VL - SEQ ID NO:9; Idi-1 VH - SEQ ID
NO:10), Idi-2 (Idi-2 VL - SEQ ID NO:11; Idi-2 VH - SEQ ID NO:12) and PAb-421
are presented in Figure 7. The CDR sequences of Idi-1 and Idi-2 are indicated
in
Figure 7and listed, along with their corresponding SEQ ID NOS, in Table 4
below:
Table 4: CDR sequences of Idi-1 and Idi-2
SEQ ID NO: Description Amino acid sequence
13 Idi-1 VL CDR1 RQSLLYKNGKTYLN
14 Idi-1 VL CDR2 LMSIRAS
Idi-1 VL CDR3 QQLVEYPYT
16 Idi-1 VH CDR1 KASGYIFTSY WIN
17 Idi-1 VH CDR2 NISPADSSTNYN
18 Idi-1 VH CDR3 EEVRRRRDMDF
19 Idi-2 VL CDR1 QASESVSFAGTSLMH
Idi-2 VL CDR2 RASKLES
21 Idi-2 VL CDR3 MQSMEDPYT
22 Idi-2 VH CDR1 KASGYSFTGYTIN
23 Idi-2 VH CDR2 LINPYNGGTCYN
24 Idi-2 VH CDR3 RVWLRRDGFYYAMDY
While the present invention has been particularly described, persons skilled
in
the art will appreciate that many variations and modifications can be made.
Therefore,
the invention is not to be construed as restricted to the particularly
described
15 embodiments, rather the scope and concept of the invention will be more
readily
understood by reference to the claims which follow.
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38

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